PAM-independent in vitro nucleic acid detection composition and detection method

Abstract

This application discloses an in vitro nucleic acid detection composition independent of the PAM motif, comprising the following components configured based on a target sequence: a Cas12a nuclease-crRNA complex capable of binding to the target sequence; and a reporter molecule capable of being trans-cleaved by the Cas12a nuclease. This application also provides a detection method: obtaining a nucleic acid amplified sample to be tested; pretreating the nucleic acid amplified sample to obtain a single-stranded sample; incubating the sample with the Cas12a nuclease-crRNA complex capable of binding to the target sequence and the reporter molecule capable of being trans-cleaved by the Cas12a nuclease; and detecting the fluorescence value of the incubated sample. The introduction of an effector capable of altering the structural state of the target sequence in this application enhances the recognition and detection signal of Cas12a-crRNA at PAM-free sites. Experimental results show that this application can specifically, flexibly, and efficiently detect the target sequence in the sample.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and more specifically, to an in vitro nucleic acid detection composition and detection method that is independent of the PAM motif. Background Technology

[0002] In vitro detection methods based on CRISPR gene editing technology offer advantages such as mild reaction temperatures, ease of operation, high specificity, and high signal amplification efficiency, overcoming the disadvantages of traditional detection methods that are typically time-consuming and require complex laboratory equipment. However, current conventional CRISPR-based detection systems are generally considered to require the presence of a PAM motif at the detection site for detection. Therefore, the development of such systems often focuses on those requiring a PAM motif (Protospacer Adjacent Motif), or on using various methods to transform sites that do not meet the PAM motif requirement into sites that do, such as developing Cas12 proteins that recognize looser PAM motifs, or adding PAM motifs to the target region through asymmetric amplification reactions. While these approaches have practical applications, they often come with side effects such as reduced detection efficiency, complex system design, and the need for extensive testing. Summary of the Invention

[0003] This application addresses the shortcomings of existing methods by proposing an in vitro nucleic acid detection composition and method that does not rely on PAM motifs, thereby solving the technical problems of insufficient specificity, flexibility, and efficiency in related technologies.

[0004] In a first aspect, this application provides an in vitro nucleic acid detection composition independent of PAM motifs, wherein the following components are configured based on a target sequence: Cas12a nuclease-crRNA complex capable of binding to the target sequence; A reporter molecule that can be trans-cleaved by the Cas12a nuclease.

[0005] Alternatively, the reporter molecule may include an ssDNA molecule with a fluorescent group and a quencher group modified at both ends, respectively, and capable of forming a hairpin structure.

[0006] Furthermore, it also includes an effector capable of altering the structural state of the target sequence, the effector being used to maintain the target sequence in a single-stranded state.

[0007] Alternatively, the effector may include a DNA oligo molecule or a single-stranded DNA-binding protein.

[0008] Further alternatively, the DNA oligo molecule comprises a pair of 50-60 nt deoxyribonucleotides, with the 5' end capable of forming a continuous hairpin structure and the 3' end capable of binding upstream or downstream of the target sequence; the single-stranded DNA binding protein comprises a single-stranded DNA molecule capable of binding to the target sequence.

[0009] Furthermore, the composition also includes components for realizing nucleic acid amplification technology.

[0010] Alternatively, the nucleic acid amplification technology may include polymerase chain reaction (PCR) or isothermal amplification.

[0011] Alternatively, the composition is suitable for in vitro detection of at least one target sequence in rice genome, human cells, and prokaryotic cells.

[0012] Secondly, this application also provides an in vitro nucleic acid detection method that does not rely on PAM motifs, which includes the following steps: Obtain the nucleic acid amplified product to be tested; The nucleic acid amplified product is pretreated to obtain a single-stranded sample for testing; The sample to be tested was incubated with a Cas12a nuclease-crRNA complex that can bind to the target sequence and a reporter molecule that can be trans-cleaved by the Cas12a nuclease. The fluorescence value of the sample to be tested after incubation was detected.

[0013] Furthermore, after the reporter molecule interacts with the Cas12a nuclease, the visible fluorescence signal emitted after ultraviolet excitation can qualitatively determine the presence of the target sequence.

[0014] Furthermore, the nucleic acid amplified material to be tested includes a fragment recognized by the Cas12a nuclease-crRNA complex through pseudo-complementary pairing and a fragment incorporating amplification primer pairs.

[0015] Further, alternatively, the pretreatment of the nucleic acid amplifier to obtain a single-stranded sample for testing includes: denaturing the nucleic acid amplifier and effector at high temperature in a buffer solution, followed by annealing, with the annealing product directly used as the sample for testing; the effector is used to maintain the single-stranded structure of the target sequence; or, The nucleic acid amplification molecule to be tested is denatured at high temperature in a buffer solution, then annealed. The annealed product is cooled and used as the sample to be tested.

[0016] The beneficial technical effects of the technical solutions provided in this application include: The system incorporates effectors (such as DNA oligos and SSB proteins) or manipulation methods capable of altering the structural state of the target sequence, specifically converting the target sequence from a double-stranded state to a single-stranded state. This enhances the recognition and detection signal of Cas12a-crRNA at sites without PAM motifs. Experimental results show that this application can specifically, flexibly, and efficiently detect target sequences (target DNA fragments) in samples. Furthermore, validation was performed in rice cells and human cells, demonstrating its applicability in plant and animal cells.

[0017] Additional aspects and advantages of this application will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this application. Attached Figure Description

[0018] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 shows the comparison results of PAM motif sites and non-PAM motif sites in plant genomes detected by the Cas12a conventional system.

[0019] Figure 2 shows the comparison results of PAM motif sites and non-PAM motif sites in human cell genomes detected by the Cas12a standard system.

[0020] Figure 3 is a schematic diagram of the detection principle of this application.

[0021] Figure 4 is a schematic diagram of the DNA Oligo molecular design of this application.

[0022] Figure 5 is a schematic diagram of the binding of the SSB protein to the target site DNA of this application.

[0023] Figure 6 shows the test results of the DNA Oligo molecule and SSB parameter design sites of this application using the rice OsEPSPS gene as the target sequence.

[0024] Figure 7 Test results of DNA Oligo molecules and SSB parameter-designed sites in this application using the rice OsDEP1 gene as the target sequence.

[0025] Figure 8 shows the comparison results of different target sequences detected using this application.

[0026] Figure 9 This is a comparison of the detection results before and after annealing of different target sequences using the annealing method without effector as described in this application.

[0027] Figure 10 The results are from testing gene-edited rice using the optimized final detection system described in this application. Detailed Implementation

[0028] The embodiments of this application are described below with reference to the accompanying drawings. It should be understood that the embodiments described below with reference to the accompanying drawings are exemplary descriptions for explaining the technical solutions of the embodiments of this application, and do not constitute a limitation on the technical solutions of the embodiments of this application.

[0029] Those skilled in the art will understand that, unless specifically stated otherwise, the terms "described" and "the" as used herein may also include plural forms. It should be further understood that the term "comprising" as used in the specification of this application means the presence of the stated features, integers, steps, operations, elements, and / or components, but does not exclude other features, information, data, steps, operations, elements, components, and / or combinations thereof supported by the art. The term "and / or" as used herein refers to at least one of the items defined by the term; for example, "A and / or B" can be implemented as "A," or as "B," or as "A and B."

[0030] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0031] First, let's introduce and explain several terms used in this application: The Cas12a nuclease-crRNA complex is a protein-nucleic acid complex that combines the characteristics of both Cas12a nuclease and crRNA. Cas12a nuclease, or Cas12a protein (also known as Cpf1), is an important endonuclease in the CRISPR-Cas system, belonging to the class Il type V system. Guided by crRNA (CRISPR RNA), Cas12a nuclease precisely recognizes the PAM sequence (TTTN, where N is any base) on target double-stranded DNA (dSDNA). TTTN sequences are widely distributed throughout the genome, theoretically covering more potential targets. However, in practical use, it exhibits cell type dependence, a high risk of off-target effects, and cannot meet the needs of many application scenarios.

[0032] DNA oligomolecules are short-chain DNA molecules, typically composed of 10-50 nucleotides linked by phosphodiester bonds. They feature a precisely designed base sequence that allows for the specific binding or amplification of target nucleic acid fragments.

[0033] Single-stranded DNA-binding proteins (SSBs / SSBPs) are a class of proteins that specifically bind to single-stranded DNA (ssDNA) and play a central role in key processes such as DNA replication, recombination, and repair. Their core functions include protecting single-stranded DNA, stabilizing its structure, and promoting the binding of related proteins.

[0034] Reporter molecules are a class of tool molecules that play an important role in the fields of biochemistry and molecular biology. Their core function is to convert the presence, location, or expression of target molecules (such as proteins and nucleic acids) into observable signals (such as fluorescence and enzyme activity) by specifically binding to or fusing with target molecules, thereby enabling the detection, tracing, or quantitative analysis of target molecules.

[0035] Polymerase chain reaction (PCR) controls the denaturation, annealing, and extension of DNA through temperature cycling, utilizing thermostable DNA polymerases (such as Tagase) to catalyze the synthesis of new strands, achieving exponential amplification of target DNA. Its core steps include: Denaturation: heating to 94-98°C to unwind double-stranded DNA into single strands; Annealing: cooling to 50-65°C, allowing primers (short single-stranded DNA) to bind to complementary regions of the single-stranded DNA; Extension: heating to 72°C, where DNA polymerase uses dNTPs as feedstock to synthesize new strands along the template strand. Each cycle doubles the amount of target DNA, and after 25-40 cycles, millions to billions of copies of a specific DNA fragment can be obtained.

[0036] Isothermal amplification technology eliminates the reliance on precise temperature control instruments, enabling rapid amplification of nucleic acids at a constant temperature through specific enzyme and primer design. Its core principle is to utilize strand displacement DNA polymerases (such as Bst enzymes) or recombinases to recognize specific regions of the target gene, forming stem-loop structures or amplifying large quantities of nucleic acid products through strand displacement reactions.

[0037] Annealing is a core step in molecular biology, referring to the process by which two complementary single-stranded nucleic acids (DNA or RNA) recombine to form a double-stranded structure under specific conditions (such as decreasing temperature) through base pairing principles (AT / U, GC). This reaction plays a crucial role in technologies such as DNA replication, PCR amplification, gene cloning, nucleic acid hybridization, and gene editing. The essence of annealing is thermodynamically driven binding between nucleic acid strands, and its core functions include: forming a double-stranded structure, providing a slab for subsequent enzymatic reactions (such as DNA polymerase extension and restriction endonuclease cleavage); specific recognition, ensuring accurate binding of the target sequence through complementary base pairing and avoiding non-specific hybridization; and signal generation, in nucleic acid hybridization techniques, the double strands formed after annealing can be detected by fluorescence, chemiluminescence, or electrochemical signals.

[0038] This application includes a PAM-independent in vitro detection system based on the Cas12a protein. The system consists of the following components: (1) a Cas12a nuclease and a crRNA molecule that binds to the Cas12a nuclease and guides it to cleave at a target sequence, thereby forming a Cas12a nuclease-crRNA complex, wherein the PAM sequence is not considered when selecting the target sequence of the crRNA, and the trans-cleavage effect of the Cas12a nuclease is initiated after recognizing the target gene sequence, non-specifically cleaving single-stranded DNA molecules in the system; (2) a reporter molecule with functional groups modified at both ends. For example, an ssDNA molecule that can form a hairpin by having a fluorescent group modified at the 5' end and a quenching group modified at the 3' end can serve as a substrate for Cas12a trans-cleavage and generate a fluorescent signal after being cleaved; (3) one or more possible tested components that can be used to pre-treat the target gene to improve the target gene detection signal level, such as DNA oligo molecules and / or single-stranded DNA binding proteins; (4) additional isothermal amplification system components required in a one-tube reaction.

[0039] The components of this application that preprocess the target sequence are mainly effectors used to change the structural state of the target sequence. For example, a pair of single-stranded DNA molecules (Oligo) about 50-60 nt long, consisting of a continuous hairpin structure at the 5' end and a single-stranded portion at the 3' end that can bind to the target gene sequence, can invade and interfere with the double-stranded structure of the target gene through annealing or other treatments; it also includes a single-stranded DNA binding protein (SSB) used to interfere with the double-stranded structure of the target gene.

[0040] Cas12a nuclease and crRNA molecules that bind to Cas12a nuclease and guide it to cleave at target sequences. When selecting the target sequence of crRNA, the PAM sequence does not need to be considered. After recognizing the target gene sequence, it can initiate the trans-cleavage effect of Cas12a nuclease and non-specifically cleave single-stranded DNA molecules in the system. ssDNA molecules with a fluorescent group modified at the 5' end and a quencher group modified at the 3' end can form hairpins and can serve as substrates for Cas12a trans-cleavage, generating a fluorescent signal after being cleaved.

[0041] This system first amplifies the target fragment in the sample using conventional polymerase chain reaction (PCR) or isothermal amplification methods, depending on the specific requirements. Then, the aforementioned paired Oligo molecules are annealed and co-incubated with the amplified target fragment, invading the double-stranded structure of the target gene and exposing the area near the recognition site as a single strand. Subsequently, under suitable buffer conditions, SSB, crRNA, and Cas12a nuclease are added to the annealed system and co-incubated, allowing the crRNA to bind to the target site according to the given spacer sequence on the crRNA and activating the nuclease's flanking cleavage effect. Once activated, the nuclease non-specifically cleaves the DNA reporter molecule in the system, producing a fluorescent signal visible to the naked eye under UV light, indicating the presence of the target DNA fragment in the sample. Our experimental results show that this in vitro detection system can specifically, flexibly, and efficiently detect the target DNA fragment in a sample.

[0042] Materials and Methods 1. Materials The crRNA, various DNA reporter molecules, and LbCas12a prokaryotic expression vector used in the experiment were all carried by Pseudomonas aeruginosa. p.aeruginosa The vectors for the target gene fragments were all artificially synthesized. For details, please refer to the table below and the sequence listing.

[0043] Table 1. Serial names and corresponding serial numbers used in this application

[0044] The SSB protein used in the experiment was E. coli Single-Stranded DNA Binding Protein.

[0045] The rice genomic DNA was obtained from the leaves of rice variety Zhonghua 11 (84-213), and the specific gene information is shown in the table above.

[0046] 2. Acquisition of target DNA fragments For each gene locus in rice, primers were designed based on the selected crRNA positions on the genome, with approximately 500 bp upstream and downstream fragments, and the fragments were amplified by PCR. The PCR product was purified, and the nucleic acid concentration was measured; this product is the detection material for the target genes in this system. The sequences of each target gene and crRNA are detailed in the sequence listing.

[0047] The non-target gene DNA material is Pseudomonas aeruginosa ( p.aeruginosaAfter the target gene fragment is synthesized and returned by a third-party company, primers are designed from a randomly selected fragment of approximately 500 bp, and the fragment is amplified by PCR. The PCR product is then purified, and the nucleic acid concentration is measured, which serves as the non-target gene DNA detection material for this system.

[0048] 3. Expression and purification of LbCas12a protein (1) The LbCas12a-6xHis prokaryotic expression vector was transformed into competent Escherichia coli BL21(DE3). Positive clones were picked and cultured in Luria-Bertani (LB) medium (containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride) at 37°C with shaking at 200 rpm.

[0049] (2) Determination of bacterial culture OD 600nm When the absorbance reaches approximately 0.6, add isopropylthio-β-galactopyranoside (IPTG) to a final concentration of 0.5 mM, and incubate at 21°C with shaking at 200 rpm for 16 h. (3) Collect the bacterial cells by centrifugation at 4℃, 4000 rpm for 20 min, add 100 ml of lysis buffer (20 mM Tris (pH 7.5), 500 mM NaCl, 10 mM mercaptoethanol, 10% glycerol, 10 mM imidazole) and 1 ml of protease inhibitor, and resuspend the bacterial pellet.

[0050] (4) The suspended bacterial cells were lysed by ultrasound in an ice bath. The ultrasound conditions were: 500W, ultrasound on for 1s, off for 1.5s, for 30min.

[0051] (5) After the bacterial cells become transparent, dispense them into 50 ml centrifuge tubes, centrifuge at 15000g for 20 min, and remove the precipitate (the precipitate and supernatant should be sampled into 1.5 ml centrifuge tubes respectively).

[0052] (6) Add 1 ml of Ni-NTA-tagged protein purification medium to the supernatant and incubate at 4°C for 1-2 h.

[0053] (7) After incubation, collect the medium into a gravity empty column and add 1 empty column volume of lysis buffer, 3 empty column volumes of high-salt buffer (20 mM Tris (pH 7.5), 1 M NaCl, 10 mM mercaptoethanol, 10% glycerol, 10 mM imidazole), 1 empty column volume of lysis buffer, and flow through the washing medium.

[0054] (8) Add an appropriate amount of elution buffer (20 mM Tris (pH 7.5), 500 mM NaCl, 10 mM mercaptoethanol, 10% glycerol, 500 mM imidazole), collect the effluent, determine the protein concentration, and detect the bands by SDS-PAGE electrophoresis.

[0055] (9) Add the effluent obtained in step 8 to the dialysis membrane (RC membrane, molecular weight cutoff of 3 kDa), tie the openings at both ends, and place it in the storage solution overnight at 4°C to replace the buffer.

[0056] (10) SDS-PAGE gel electrophoresis identification and preservation.

[0057] 4. In vitro detection reaction of Cas12a and crRNA Prepare a reaction mixture of LbCas12a (200 nM) and crRNA (250 nM) in NEB Buffer 3.1 and incubate at 37°C for 30 min to form a complex. Then, add 500 nM DNA fluorescent reporter molecule and 10 nM target gene fragment to the 2.5 μL complex to make a 10 μL reaction mixture and incubate at 37°C for 1 h. Read the fluorescence value after incubation.

[0058] 5. DNA oligo annealing pretreatment and PAM-free detection system based on Cas12a-crRNA with added SSB protein. Method 1: Mix 10 nM target gene, 1 μM DNA oligo, and annealing buffer. Incubate at 95°C for 2 min to fully denature the oligo, then decrease the temperature by 10°C every 1 min for annealing. The annealed product is used directly as the target gene. Prepare a reaction mixture of LbCas12a (200 nM), crRNA (250 nM), and SSB (10 μg / μL) in NEBBuffer 3.1 and incubate at 37°C for 30 min to form a complex. Take 2.5 μL of the complex, 3 μL of the annealed product, and 500 nM DNA fluorescent reporter molecule, mix to form a 10 μL reaction mixture, incubate at 37°C for 30 min-1 h, and then read the fluorescence value.

[0059] Method 2: 10 nM target gene and annealing buffer were added and incubated at 95°C for 2 min to fully denature the target gene fragment. After annealing, the fragment was placed on ice to cool. The annealed product was used directly as the target gene. LbCas12a (200 nM) and crRNA (250 nM) were prepared in NEB Buffer 3.1 and incubated at 37°C for 30 min to form a complex. 2.5 μL of the complex, 3 μL of the annealed product, and 500 nM of DNA fluorescent reporter molecule were mixed to form a 10 μL reaction system. The mixture was incubated at 37°C for 30 min-1 h, and the fluorescence value was then read.

[0060] Example 1: The Cas12a conventional detection system is partially detectable for non-PAM motif sites.

[0061] To assess the effectiveness of Cas12a in detecting non-PAM motif sites, we selected 18 non-PAM motif sites from plant and human cell genomes and designed crRNA for detection. The results showed that Cas12a could detect 12 of the selected sites, and the signal values ​​of the target gene experimental group were significantly different from the control background. However, the signal levels of 4 of these sites were relatively low; 6 sites could not produce a signal or only produced a very weak signal in the presence of high concentrations of the target gene fragment. Figure 1 and Figure 2 ).

[0062] Example 2: Preprocessing the target gene fragment can improve the detection level of Cas12a for non-PAM motif sites.

[0063] Our experimental results show that about one-third of the non-PAM motif sites can only produce very weak signal values ​​when the target gene fragment is present at a high concentration. In order to improve the detection signal of these inefficient sites, we optimized the detection system. Based on the published research results, we speculate that the target site in the single-stranded state can be better recognized by the Cas12a-crRNA complex and trigger the trans-cleavage activity of Cas12a. In order to expose the target gene site and maintain the single-stranded state, we pretreated the target gene fragment using the following two components: (1) DNA oligo that can complement the target gene and has a stable hairpin structure; (2) single-stranded DNA binding protein (i.e., SSB). Among them, DNA oligo can invade the double-stranded structure of the target site DNA by complementarizing with the target site. Previous studies have shown that SSB can specifically bind to the single strand of DNA in the system, while destabilizing the bound DNA double-stranded structure. Figures 3-5 ).

[0064] Regarding specific design parameters, we designed corresponding oligo molecule pairs around the crRNA recognition region, with upstream and downstream distances of 50 bp, 100 bp, and 150 bp for the target gene region, and made different combinations of the distribution of oligo molecule pairs on the target and non-target chains. Figures 6-7 (and Table 1).

[0065] Example 3: Testing and optimization of DNA oligo molecules and SSB design parameters.

[0066] As in Example 2 ( Figures 6-7 As shown in the rice genome... OsEPSPS and OsDEP1 Detection was performed at two PAM-free motif sites. With the target gene concentration in each group at 10 nM, the experimental results showed that: (1) in the conventional detection system without the introduction of DNA oligo and SSB treatment, OsEPSPS and OsDEP1 The low signal levels at both sites indicate that these sites are difficult to detect effectively by Cas12a-crRNA in the absence of PAM sequences. (2) The introduction of DNA oligos significantly enhances the detection signal values ​​at these two sites, with an enhancement of approximately 4 times. Among them, the design with a distance of 150 bp upstream and downstream of the recognition site and distributed on the target and non-target chains respectively shows a more significant enhancement effect between sites. (3) The addition of SSB to the system can generally enhance the signals of all groups treated with DNA oligos, with an increase of more than 30%. Figure 6 and Figure 7 ).

[0067] In summary, the introduction of DNA oligo and SSB protein into the system can enhance the recognition and detection signals of Cas12a-crRNA for sites without PAM motifs.

[0068] Example 4: Detection of PAMless sites on plant and animal genomes using the Cas12a detection system pretreated with DNA Oligo and SSB.

[0069] Based on the results of Example 3, we selected the DNA oligo design parameters of Oligo-150bp-2 and the detection conditions such as SSB protein as our optimized system 1 to detect PAM-free motif sites on plant and animal genomes.

[0070] The specific detection conditions are as follows: Before detection, we annealed Oligo molecules (final concentration 1 μM) with the target gene in annealing buffer. Then, we incubated the annealed product, Cas12a-crRNA complex (final concentration 50 nM), and SSB protein (total concentration 10 μg / μL) in a reaction buffer system for 30 min–1 h. After the reaction, we measured the fluorescence signal value.

[0071] Experimental results are as follows Figure 8 The results showed that: (1) Of the 17 loci detected in the plant and animal genomes, the detection levels of 9 loci were significantly improved after treatment with DNA oligo molecules and SSB protein at the same target gene concentration; the detection levels of 6 loci remained similar at the same concentration after treatment with DNA oligo molecules and SSB protein; and the detection levels of 2 loci showed a slight decrease after treatment, but this did not affect the overall detection trend. (2) In OsEPSPS and hHEK3 At sites that are difficult or weak to detect using conventional systems, the detection signal is significantly enhanced after treatment with DNA oligo molecules and SSB protein, up to approximately 8 times.

[0072] These results show that the optimized system 1, which incorporates DNA oligo molecules and SSB protein for pretreatment, can significantly improve the recognition and response level of Cas12a-crRNA to PAM-free motif target sites.

[0073] Example 5: The Cas12a detection system, which annealed the target gene fragment, was used to detect PAMless sites on the genomes of plants and animals.

[0074] The above experimental results show that target sites in a single-stranded state are better recognized by the Cas12a-crRNA complex and trigger the trans-cleavage activity of Cas12a. Therefore, in addition to using DNA Oligo and SSB to pretreat the target gene fragment to make it single-stranded, we also used a high-temperature annealing method to maintain the target gene fragment in a single-stranded state and detected it in the absence of PAM motif sites on the plant genome.

[0075] The specific detection conditions are as follows: Before detection, we annealed the target gene fragment to a final concentration of 10 nM in annealing buffer at 95°C for 2 minutes. After annealing, we immediately removed it and placed it on ice to cool. Then, we co-incubated 3 μL of the annealing product, 50 nM of the Cas12a-crRNA complex, and 500 nM of the DNA fluorescent reporter molecule in the reaction buffer system for 30 min–1 h. After the reaction, we measured the fluorescence signal value. The experimental results are as follows: Figure 9 As shown, four out of the six sites we examined exhibited similar levels of fluorescence signal before and after annealing. This was evident in direct observation of... OsAAT and OsEPSPS When these two sites were detected, the fluorescence signal was very weak. Further detection after annealing the target gene fragment revealed… OsAAT The fluorescence signal at the site increased by about 10 times. OsEPSPS The site fluorescence signal increased by about 5 times.

[0076] The experimental results above show that annealing the target gene fragment can improve the detection level of PAM less sites in the Cas12a-crRNA detection system and ensure stable detection of the target gene.

[0077] Example 6: Based on the above experimental results, we used a method of pre-annealing the target gene fragments in rice plants to detect gene editing. We tested three rice leaf genes: OsGAPDH-1, OsACC1-1, and OsEPSPS-1. The experimental results are as follows: Figure 10 As shown, despite the absence of a suitable PAM sequence prior to the editing site, the method used in this embodiment can accurately detect the edited mutant plants. The experimental results are completely consistent with the genotypes of each plant (determined by sequencing), demonstrating the accuracy and sensitivity of the method.

[0078] In summary, this application provides a PAM-independent in vitro nucleic acid detection composition based on the following components configured according to the target sequence: a Cas12a nuclease-crRNA complex capable of binding to the target sequence; and a reporter molecule capable of being trans-cleaved by the Cas12a nuclease. This application also provides a detection method: obtaining a nucleic acid amplified sample to be tested; pretreating the nucleic acid amplified sample to obtain a single-stranded sample; incubating the sample with the Cas12a nuclease-crRNA complex capable of binding to the target sequence and the reporter molecule capable of being trans-cleaved by the Cas12a nuclease; and detecting the fluorescence value of the incubated sample. The introduction of an effector capable of altering the structural state of the target sequence in this application enhances the recognition and detection signal of Cas12a-crRNA for PAM-free sites. Experimental results show that this application can specifically, flexibly, and efficiently detect the target sequence in the sample.

[0079] This invention relates to a series of pretreatments, with or without the methods described below (such as adding short DNA molecules complementary to the target gene, adding DNA single-strand binding proteins, etc.), for in vitro detection of nucleic acid fragments in samples by binding to the Cas12a-crRNA protein complex, and generating a detection signal via a DNA reporter molecule carrying a fluorescently modified group. This system can be used for the detection of a wide range of exogenous nucleic acid fragments or gene mutations.

[0080] Those skilled in the art will understand that the steps, measures, and solutions in the various operations, methods, and processes discussed in this application can be alternated, modified, combined, or deleted. Furthermore, other steps, measures, and solutions in the various operations, methods, and processes discussed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted. Furthermore, steps, measures, and solutions in related technologies that are similar to those disclosed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted.

[0081] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0082] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0083] The above description is only a partial implementation of this application. It should be noted that for those skilled in the art, other similar implementation methods based on the technical concept of this application, without departing from the technical concept of this application, also fall within the protection scope of the embodiments of this application.

Claims

1. A PAM-independent in vitro nucleic acid detection composition, characterized in that, The following components are configured based on the target sequence: Cas12a nuclease-crRNA complex capable of binding to the target sequence; A reporter molecule that can be trans-cleaved by the Cas12a nuclease.

2. The PAM-independent in vitro nucleic acid detection composition as described in claim 1, characterized in that, The reporter molecule includes an ssDNA molecule with a fluorescent group and a quencher group modified at both ends, respectively, and capable of forming a hairpin structure.

3. The PAM-independent in vitro nucleic acid detection composition as described in claim 1, characterized in that, It also includes an effector capable of altering the structural state of the target sequence, the effector being used to maintain the target sequence in a single-stranded state.

4. The PAM-independent in vitro nucleic acid detection composition as described in claim 3, characterized in that, The effectors include DNA oligo molecules or single-stranded DNA-binding proteins.

5. The PAM-independent in vitro nucleic acid detection composition as described in claim 4, characterized in that, The DNAoligo molecule comprises a pair of 50-60 nt deoxyribonucleotides, with the 5' end capable of forming a continuous hairpin structure and the 3' end capable of binding upstream or downstream of the target sequence; the single-stranded DNA binding protein comprises a single-stranded DNA molecule capable of binding to the target sequence.

6. The PAM-independent in vitro nucleic acid detection composition as described in claim 1, characterized in that, The composition also includes components for implementing nucleic acid amplification technology.

7. The PAM-independent in vitro nucleic acid detection composition as described in claim 6, characterized in that, The nucleic acid amplification technology includes polymerase chain reaction (PCR) or isothermal amplification.

8. The PAM-independent in vitro nucleic acid detection composition as described in claim 1, characterized in that, The composition is suitable for in vitro detection of at least one target sequence from rice genome, human cells, and prokaryotic cells.

9. A PAM-independent in vitro nucleic acid detection method, characterized in that, It includes the following steps: Obtain the nucleic acid amplified product to be tested; The nucleic acid amplified product is pretreated to obtain a single-stranded sample for testing; The sample to be tested was incubated with a Cas12a nuclease-crRNA complex that can bind to the target sequence and a reporter molecule that can be trans-cleaved by the Cas12a nuclease. The fluorescence value of the sample to be tested after incubation was detected.

10. The method as described in claim 9, characterized in that, After the reporter molecule interacts with the Cas12a nuclease, the visible fluorescence signal emitted after ultraviolet excitation can qualitatively determine the presence of the target sequence.

11. The method as described in claim 9, characterized in that, The nucleic acid amplified material to be tested includes a fragment recognized by the Cas12a nuclease-crRNA complex through pseudo-complementary pairing and a fragment incorporating amplification primer pairs.

12. The method as described in claim 9, characterized in that, The pretreatment of the nucleic acid amplifier to obtain a single-stranded sample for testing includes: denaturing the nucleic acid amplifier and effector in a buffer solution at high temperature, followed by annealing, with the annealed product directly used as the sample for testing; the effector is used to maintain the single-stranded structure of the target sequence; or... The nucleic acid amplification molecule to be tested is denatured at high temperature in a buffer solution, then annealed. The annealed product is cooled and used as the sample to be tested.

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